Difference between revisions of "Part:BBa K2933212"
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===Molecular cloning=== | ===Molecular cloning=== | ||
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'''Figure 1.''' The result of PCR | '''Figure 1.''' The result of PCR | ||
Revision as of 12:57, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+GIM-2+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+GIM-2），and the biological module can be built into E.coli for protein expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 47
Illegal PstI site found at 935 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
Illegal PstI site found at 935 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 935 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 935 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology=
This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS and our target protein GIM-2. It encodes a protein which is GIM-2 fused with His tag. The fusion protein is about 27.4 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of GIM-2. It is convenient for us to purify our target protein.
Molecular cloning
Figure 1. The result of PCR
References
1. Skagseth S , Akhter S , Paulsen M H , et al. Metallo-β-lactamase inhibitors by bioisosteric replacement: Preparation, activity and binding[J]. European Journal of Medicinal Chemistry, 2017, 135:159-173.
2. Wendel AF, MacKenzie CR. 2015. Characterization of a novel metallo-lactamase variant, GIM-2, from a clinical isolate of Enterobacter cloacae in Germany. Antimicrob Agents Chemother 59:1824 –1825.
3. Borra P S , Samuelsen O , Spencer J , et al. Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-beta-Lactamases[J]. Antimicrobial Agents and Chemotherapy, 2013, 57(2):848-854.
4. Susann S, Trine J C, Gro Elin K B, James S, Ørjan S, Hanna-Kirsti S. L. Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1. Antimicrobial Agents and Chemotherapy Jan 2016, 60 (2) 990-1002